1. Field of the Invention
This invention relates to the field of cell biology and in particular to carriers useful for growing attachment-dependent cells in vitro.
2. Prior Art
There has long been interest in the growth of primary, human diploid and other attachment-dependent cells in large quantities. Such interest is intensified as the demand for cells and cell by-products for research and commerical applications has increased. Many techniques have been developed for large scale production of cells. Typical of these developments are roller bottles, multiplate and spiral film propagators, hollow fiber and glass helix perfusion systems. Unfortunately, these techniques are generally cumbersome and pose inherent problems of cell manipulation and observation, media perfusion, batch homogeneity and scale-up.
More recently an alternate technology for mass cell culture has been developed which overcomes these problems. The original experiments by van Wezel, Nature 216, 64 (1967) employed charged dextran beads for culture of established cell lines, human diploid cells and primary rabbit kidney cells. Subsequent work by van Wezel, "Microcarrier Culture of Animal Cells" in Tissue Culture Methods and Applications, p. 372 (Kruse and Patterson, Eds.) Academic Press, NY (1973) and others, e.g. Horng and McLimans, Biotechnol. Bioeng., 17, 713 (1975), centered on eliminating the cytotoxic effects of these beads and on better defining the necessary characteristics of microspheres suitable for cell culture. Finally, Thilly and Levine established the importance of bead charge density, (Levine et al., Somatic Cell Genetics, 3, 149 (1977)) determining 150 .mu.m diameter beads carrying 2 meq/g charge density as optimal for cell attachment and proliferation and in U.S. Pat. No. 4,036,693 teach a method for treating derivatized dextran beads. As a result, specially treated beads have been optimized for cell culture and systems have been adapted for the routine production of large quantities of cells and viral vaccines (Giard, et al., Applied and Environ Microbiol, 34, 668 (1977). van Hemert, Biotechnol Bioeng., 6, 381 (1964). Spier, et al., Biotechnol Bioeng., 19, 1735 (1977)).
Dextran and derivatized dextran beads, although known and used as culture carriers, nevertheless have problems associated with their use. They are not known to be impervious to attack by enzymes or bacteria. Most importantly, toxic effects, especially the initial cell destruction, have been observed. While early work of Thilly and Levine appeared to explain this as a function of too much DEAE functionality density, it appears that other parameters, less easily defined and controlled are involved, (van Wezel et al., Process Biochem, 3, 6-8, 28 (1978)). Moreover, preparation of dextran-based beads is a multi-step process. Typically, charged dextran particles are made by first producing a dextran bead and then reacting it with a charged group such as DEAE to form the end product. Charged dextran may then be further treated, such as is taught in U.S. Pat. No. 4,036,693, in attempting to control toxic effects. The need for a totally new microcarrier has been recognized, (van Wezel et al., id., p. 8). Many dimethylaminopropylmethacrylamide polymers and co-polymers have been developed and are known as useful ion exchange resins. U.S. Pat. Nos. 2,567,836 and 3,287,305 disclose typical examples of this class of copolymers and known methods of their preparation.